Sperm class analyzer

Semen samples were collected from four healthy, fertile men. After retrieval, the semen samples were liquefied at a constant temperature of 37°C for 30–60min. After liquefaction, sperm parameters were determined using a computer-assisted sperm analysis system (Sperm Class Analyzer; Microptic, Barcelona, Spain). The spermatozoa were purified by gradient centrifugation using 90% and 45%gradients (SpermGrad; Vitrolife, Kungsbacka, Sweden) at 300 × g for 20min.The resulting supernatant was removed, and the sperm pellets were washed twice with G-IVF medium(G-IVF; Vitrolife, Kungsbacka, Sweden). Finally, the spermatozoa were cultured in G-IVF medium at a concentration of approximately 10 × 10⁶/mL.

Semen analysis was performed in the laboratory department of our center according to the fifth edition of World Health Organization (WHO) guidelines [3 (link)]. Specifically, the normal range of spermatozoa parameters were as follows: semen volume, ≥ 1.5 mL; sperm concentration, ≥ 15 million/mL; total sperm motility, ≥ 40%; normal morphology of spermatozoa, ≥ 4% [3 (link)]. The number, motility and morphology of spermatozoa were assessed through a computer-aided spermatozoa analysis system (Sperm Class Analyzer, MICROPTIC, Barcelona, Spain). The intra- and inter-assay coefficients of variation were both set to < 15%.

Sperm motility was quantified using a computer-assisted sperm analysis system (CASA, Sperm Class Analyzer®, Microptic, Barcelona, Spain). Briefly, a sperm sample (2 μl) was placed in a pre-warmed (38 °C) Leja counting slide (Leja Products B.V., Nieuw-Vennep, The Netherlands), and 10 fields were analyzed at 38 °C to assess a minimum of 1000 spermatozoa per sample for total motile sperm (%) and progressive motile sperm (%).

For sperm sampling, the rat cauda epididymis was immediately washed in PBS after removal, and placed in DMEM/Ham’s F-12 at 37°C. The cauda epididymis was cut into small pieces in a Petri dish containing the same medium to remove blood vessels and fat tissue. Half a centimeter of the cauda epididymis was transferred to another Petri dish containing 1 mL medium, and the sperm was allowed to swim up for 1 min.
Five microliters of the sperm suspension was loaded into a Leja slide (Leja Products BV, Nieuw Vennep, Netherlands). A 4× negative phase contrast objective combined with a phase contrast condenser was used to determine sperm motility and concentration via the Motility/Concentration module of the Sperm Class Analyzer^® version 5.4.0.1 software (Microptic S. L., Barcelona, Spain) at 25 frames/second. For motility analysis, 200 motile spermatozoa were analyzed as recommended [18 , 19 (link)].

Male birds were humanely euthanized in accordance with Schedule 1. Live sperm were collected within 20 min from the left seminal glomerus (SG) and analysed using the Sperm Class Analyzer^® (Microptic, Barcelona, Spain), following methods described in [23 (link)] (electronic supplementary material).
For the 2015 cohort, the remaining mature sperm (from the distal third portion of the SG) was squeezed into 110 µl of phosphate-buffered saline (PBS) at room temperature (20°C) to avoid activating motility. The sperm suspension was thoroughly aspirated in an eppendorf tube using a pipette and 10 µl was fixed in 90 µl of 5% formalin for sperm concentration and morphology analyses at a later date (see below). The remaining 100 µl was used to quantify ATP content using an ATPlite 300 assay kit (Perkin Elmer, UK) with a modified protocol (described below) allowing sample collection and analysis to be carried out on separate days. ATP was isolated from the sperm suspension by adding 250 µl of PBS, 175 µl of mammalian cell lysis reagent (from ATPlite kit) and incubating at room temperature for 5 min, while mixing with a vortex for 10 s every minute. Samples were centrifuged at 12 000 × g for 2.5 min, and the supernatant was snap-frozen in liquid nitrogen and stored at −80°C until quantification.

Five microliters of concentrated spermatozoa cloud was collected and placed on a Leja slide (Leja Products BV, Nieuw Vennep, Netherlands). The Leja slide was placed onto a temperature-controlled stage of the Nikon E200 microscope (37°C). A 4x negative phase-contrast objective in conjunction with a phase-contrast condenser was used to determine sperm motility and concentration via the motility/concentration module of the Sperm Class Analyzer® version 5.4.0.1 software (Microptic SL, Barcelona, Spain) at 50 frames/s. Data were collected by capturing images with a digital camera (Basler, A78075gc, Germany). For motility analysis, eight fields were captured with the SCA system until 200 motile spermatozoa were analyzed, as recommended by WHO (1999) [46 (link)–48 (link), 51 (link)].